With the introduction of glutaraldehyde preservation of biological tissue, and in particular porcine bioprosthetic heart valves, it has become possible to: (a) overcome the poor performance of early formaldehyde-preserved implanted tissue valves; (b) discontinue the use of homograft valves; and (c) avoid the undesirable use of anitcoagulants required to prevent thromboembolism associated with the use of non-bioprosthetic (mechanical) heart valves, especially in children. Not unlike other similarly important discoveries, however it appears that the glutaraldehyde-preserved bioprosthesis has created its own dilemma.
Although the relatively biologically inert glutaraldehyde-preserved valves of Carpentier and others have demonstrated excellent long-term durability in most instances, serious drawbacks such as tissue-fatigue and a propensity toward calcification have occured. Moreover, it was initially contemplated that children and adolescents would be among those deriving the greatest benefit from the glutaraldehyde-preserved bioprosthetic heart valves since the anticoagulants required with mechanical prostheses could be eliminated. Results from an increasing number of recent clincial studies indicate that severe calcification of these tissues with relatively short-term failure is prevalent among children and adolescents. Thus, despite their long-term durability and overall reduced incidence of complications, these glutaraldehyde-preserved valves have been deemed by some to be unsuitable for use in children.
Calcification of tissue remains a mystery for the most part; however, it has previously been shown that various factors including calcium metabolism the present invention, porcine heart valves or pericardinal tissue which was fixed in glutaraldehyde and subsequently treated with a surfactant was implanted subcutaneously in rabbits. This treated tissue unexpectedly and beneficially effected a sustained mitigation or reduction of calcification after implantation. This sustained mitigation of calcification provides a method of increasing the durability of implanted tissue, particularly of heart valve bioprostheses.
In accordance with the present invention, the tissue may be stored and processed in conventional well-known conditions and may be fixed (tanned) conventionally in from about 0.2 to about 0.6 weight percent and preferably from about 0.5 to about 0.7 weight percent glutaraldehyde in either phosphate-buffered solutions, or phosphate-free buffers as described hereinafter. The tissue handling conditions as conventionally known are not considered part of our present invention unless otherwise stated. Likewise, tissue may be sterilized in 0.625 percent glutaraldehyde or from about 4 to about 5 percent formaldehyde.
Organic surfactants within the scope of the present invention include anionic, cationic, and nonionic surfactants and their salts. Preferred salts of the surfactants in the present invention include sodium, potassium, ammonium, and halide. Anionic surfactants of the present invention are those having a relatively large hydrophobic region of hydrocarbon residues including both aliphatic groups, aromatic groups and combinations thereof bonded to a negatively charged ionic group. The aliphatic residues may be branched chains, straight chains, cyclic, heterocyclic, saturated or unsaturated. These hydrophobic residues may either be connected directly to an anionic group such as carboxylate, sulfate, or sulfonate; or connected thereto through an intermediate linkage such as an ester, amide, sulfonamide, ether, or aryl group. Anionic surfactants in one embodiment of the present invention are those having carboxylates bonded to the alkyl side chain of a steroid or through amino acids in the side chain; such as in the bile acids. Illustrative bile acids in accordance with the present invention include but are not limited to deoxycholic acid, cholic acid, lithocholic acid, taurocholic acid, and glycocholic acid, and their salts. A preferred bile acid and its salt which we have found effective in mitigation of calcification of implanted tissue is sodium deoxycholate. Anionic surfactants in accordance with the present invention further include those having a carboxylate group bonded to a straight-chained aliphatic group preferably having from about 8 to about 20 carbon atoms; such as the sodium salts of fatty acids. Anionic surfactants containing carboxylate groups in accordance with the pesent invention further include those having the carboxylate group coupled to a hydrophobic portion through an amide, sulfonamide, or ester linkage such as in the N-alkanoyl amino acids and N-acylated amino acids. Illustrative of N-alkanoyl amino acids are those including but not limited to surfactants having the formula R.sub.1 CONR.sub.2 CHR.sub.3 CO.sub.2 -- where R.sub.1 is an aliphatic residue preferably having from about 8 to about 18 carbon atoms, R.sub.2 is hydrogen or methyl, and R.sub.3 is a conventional amino acid side chain. Illustrative side chains include the nonpolar aliphatic side chains of alanine, leucine, isoleucine, valine, and proline; the aromatic rings of phenylalanine and tryptophan; the polar side chains of glycine, serine, threonine, cystine, and the like; and the charged polar groups of aspartic acid, glutamic acid, lysine, and the like. Preferred carboxlate containing surfactants in accordance with this embodiment of the present invention are those containing an amide linkage such as N-lauroylsarcosine.
Anionic surfactants in accordance with an alternate embodiment of the present invention include ethylene oxide modified sulfates of aliphatic alcohols, sulfated ethanol amides, or alkyl phenols such as the sulfonated alkylphenyl ethers. Further anionic surfactants include alkane sulfonic acids and alkylaryl sulfonic acids. Alkane sulfonic acids in accordance with the present invention include those having the sulfur directly attached to the hydrophobic residue, such as 1-decanesulfonic acid; or coupled through an ester, amide, or ether; such as N-methyltaurine. Alkylaryl sulfonates are those having the sulfur directly attached to an aromatic ring such as phenyl or napthyl which is, in turn, coupled to the hydrophobic residue preferably having from about 8 to about 18 carbon atoms. Illustrative of this latter type of surfactant is dodecylbenzenesulfonic acid.
Cationic surfactants in accordance with the present invention include alkyl quaternary amines and their halide slats. Preferable surfactants in the present invention include the chloride and bromide salts of tertiary amines connected directly to a hydrophobic residue or connected thereto through an amide linkage. Preferably the amines are directly connected to a relatively large hydrophobic portion having an aromatic residue such as benzene, pyridine or napthylene; aliphatic chain which is branched, unbranched, cyclic, saturated, or unsaturated; or a combination of both aromatic and aliphatic residues. Illustrative alkyl quaternary ammonium surfactants include but are not limited to cetylpyridinium chloride, cetyltrimethylammonium bromide, trimethylphenylammonium chloride, decytrimethylammonium bromide, hexdecyltrimethylammonium bromide, and the like.
Nonionic surfactants in accordance with the present invention include polyoxyalkylene ethers, polyoxyalkylene alkylaryl ethers, aliphatic esters, polyethers, polyoxyalkylene ester derivatives, saccharide ester derivatives, and combinations thereof. Nonionic polyoxyalkylene, and preferably polyoxyethylene, ethers are those having a relatively long hydrophobic residue and a hydroxyl end connected by one or more alkylene oxide residues. Examples of polyoxyalkylene ethers are polyoxyethylene lauryl ether (Brij), polyoxyethylene oleyl ether, polyoxyethylene cetyl ether, and the like. Nonionic polyoxyalkylene, and preferably polyoxyethylene, alkylaryl ethers are those having a relatively large hydrophobic residue and a hydroxyl end connected thereto by an aryl, such as benzene or napthaline and one or more alkylene oxide residues. Examples of polyoxyalkylene alkylaryl ethers include polyethylene Glycol p-Isooctyl phenyl ethers such as Triton X-100 and the like. Nonionic polyethers are those having the formula CH.sub.3 (CH.sub.2).sub.N --O--(C.sub.2 H.sub.4 O).sub.M where N is about 11, and M is about 23.
Nonionic aliphatic esters include aliphatic fatty acid esters, polypropyleneglycol fatty acid esters such as propyleneglycol monostearate, and glycerol fatty acid esters such as glycerol monostearate. Aliphatic fatty acid esters are those having the formula R.sub.4 COOR.sub.5 where R.sub.4 is an alkyl preferably having from about 8 to about 20 carbon atoms, and R.sub.5 is an aliphatic residue having from 1 to about 5 carbon atoms. Saccharide and polyoxyalkylene ester derivatives are those having either a 5 or 6 carbon sugar in the former or a polyoxyalkylene chain in the latter coupled to a relatively long hydrophobic residue through an ester bond. Illustrative saccharide derivatives include sorbitol coupled to fatty acids to form surfactants such as sorbitan trioleate, sorbitan strearate, sorbitan monooleate, and the like. Polyoxyalkylene ester derivatives include polyoxyethylene monooleate, polyoxyethylene monostearate, and the like. Combinations of polyoxyalkylene ether derivatives and sorbitol ester derivatives found to be useful in the present invention include Polyoxyethylene sorbitan fatty acid derivatives such as polyoxyethylene (20) sorbitan monooleate (Polysorbate-80, Tween-80 manufactured by DIFCO).
In accordance with the present invention, the effective concentration of surfactant will vary somewhat depending on the molecular weight thereof; and is preferably from about 0.1 to about 10 percent (w/v) and more preferably from about 0.5 to about 5 percent. Most preferably, the surfactant concentration is from about 0.5 to about 1.5 percent. Moreover, the treatment of the tissue with surfactant can be performed during the fixation (tanning) process, during the sterilization process, or in a separate step after fixation and prior to sterilization; and from about 2 to about 30 hours and preferably from about 6 to about 24 hours.
In accordance with a preferred embodiment of the present invention, the tissue is treated with surfactant at temperatures of from about 20.degree. C. to about 40.degree. C. In one embodiment, the surfactant is included in the sterilization step whose effectiveness has been found to be enhanced at temperatures above room temperature (20.degree. C.) to a range of from about 30.degree. to 40.degree. C.
In accordance with the present invention, it is preferable to store, fix, and sterilize the tissue within a tissue-stabilizing pH range; that is, within a pH range that is not deleterious to the tissue components. A preferred pH range is from about 7.0 to about 7.6, and a more preferred pH range is from about 7.1 to about 7.4. The most preferred pH in accordance with the present invention is 7.3.
Buffers used in accordance with one embodiment of the present invention are preferably stable, non-interacting with the stabilization process, and have a buffering capacity sufficient to maintain an acceptable pH, particularly during the fixation of the tissue. The choice of the appropriate buffer, and its concentration will depend upon specific tissue preparation conditions; variations of which have been introduced by several manufacturers. The buffers can be either conventional 0.01-0.02M phosphate-buffered saline (PBS) or phosphate-deficient solutions such as those containing less phosphate than these 0.01 to 0.02M PBS solutions, and preferably less than about 0.001 to about 0.002M phosphate. Preferred buffers in accordance with the present invention include borate, carbonate, bicarbonate, cacodylate (found to be nontoxic in animals), and other synthetic, artificial, or organic buffers such as HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid; MOPS, 2-(N-morpholino) propane-sulfonic acid; and PIPES, 1,4-piperazinediethanesulphonic acid.
Preferably, the buffered or unbuffered solutions, used in accordance with the present invention should not interfere with the tissue stabilizing process afforded by fixing agents such as glutaraldehyde. That is, they should not react with the fixing agent or prevent the fixing agent from achieving proper fixation of the tissue. Illustrative of this are buffers containing primary and secondary amines such as tris(hydroxymethyl)aminomethane (Tris), which are known to react with the aldehyde groups of glutaraldehyde or formaldehyde and thus interfere with the normal tissue stabilization process.
The present invention is further illustrated by the following examples which are not intended to be limiting: